This project is designed to answer the longstanding question of when genetic recombination takes place during meiosis. Our approach involves physically detecting recombined DNA molecules during an ongoing, synchronous meiosis in the yeast Saccharomyces cerevisiae. Recombination is detected by the formation of new-sized EcoRl restriction fragments for the SUP4 genetic region in a diploid strain that is heterozygous for the presence of two adjacent restriction sites. These restriction fragments can be detected at less than 1% of single copy DNA by southern blotting techniques. The timing of the generation of the rare recombinant-sized fragments will be correlated with the cytological, genetical and DNA biochemical landmarks of meiosis. We will also look for restriction site polymorphisms adjacent to other regions of the genome, especially at several recombinational "hot spots". Using cloned DNA sequences at or near arg4, ade8, and MAT we will examine the timing of recombination at these sites as well. This information will help to clarify the role that various cytological structures play in meiosis. This analysis may also help explain why meiotic recombination plays an apparently essential role in proper chromosome segregation during gametogenesis. We will also examine several mutants that are either unable to complete meiosis or to produce viable haploid spores for their ability to physically carry out recombination. This will enable us to determine which mutants affect the recombination process directly. In addition, the methods developed here may make it possible to detect DNA intermediates in meiotic recombination. This would greatly improve our knowledge of the molecular mechanism of recombination.